Exploring the impact of lactic acid bacteria on the biocontrol of toxigenic Fusarium spp. and their main mycotoxins.

The occurrence of fungi and mycotoxins in foods is a serious global problem. Most of the regulated mycotoxins in food are produced by Fusarium spp. This work aimed to assess the antifungal activity of selected lactic acid bacteria (LAB) strains against the main toxigenic Fusarium spp. isolated from cereals. Various machine learning (ML) algorithms such as neural networks (NN), random forest (RF), extreme gradient boosted trees (XGBoost), and multiple linear regression (MLR), were applied to develop models able to predict the percentage of fungal growth inhibition caused by the LAB strains tested. In addition, the ability of the assayed LAB strains to reduce/inhibit the production of the main mycotoxins associated with these fungi was studied by UPLC-MS/MS. All assays were performed at 20, 25, and 30 °C in dual culture (LAB plus fungus) on MRS agar-cereal-based media. All factors and their interactions very significantly influenced the percentage of growth inhibition compared to controls. The efficacy of LAB strains was higher at 20 °C followed by 30 °C and 25 °C. Overall, the order of susceptibility of the fungi to LAB was F. oxysporum > F. poae = F. culmorum ≥ F. sporotrichioides > F. langsethiae > F. graminearum > F. subglutinans > F. verticillioides. In general, the most effective LAB was Leuconostoc mesenteroides ssp. mesenteroides (T3Y6b), and the least effective were Latilactobacillus sakei ssp. carnosus (T3MM1 and T3Y2). XGBoost and RF were the algorithms that produced the most accurate predicting models of fungal growth inhibition. Mycotoxin levels were usually lower when fungal growth decreased. In the cultures of F. langsethiae treated with LAB, T-2 and HT-2 toxins were not detected except in the treatments with Pediococcus pentosaceus (M9MM5b, S11sMM1, and S1M4). These three strains of P. pentosaceus, L. mesenteroides ssp. mesenteroides (T3Y6b) and L. mesenteroides ssp. dextranicum (T2MM3) inhibited fumonisin production in cultures of F. proliferatum and F. verticillioides. In F. culmorum cultures, zearalenone production was inhibited by all LAB strains, except L. sakei ssp. carnosus (T3MM1) and Companilactobacillus farciminis (T3Y6c), whereas deoxynivalenol and 3-acetyldeoxynivalenol were only detected in cultures of L. sakei ssp. carnosus (T3MM1). The results show that an appropriate selection and use of LAB strains can be one of the most impacting tools in the control of toxigenic Fusarium spp. and their mycotoxins in food and therefore one of the most promising strategies in terms of efficiency, positive impact on the environment, food safety, food security, and international economy.

Silver Nanoparticle-Based Therapy: Can It Be Useful to Combat Multi-Drug Resistant Bacteria?

The present review focuses on the potential use of silver nanoparticles in the therapy of diseases caused by antibiotic-resistant bacteria. Such bacteria are known as "superbugs", and the most concerning species are Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus (methicillin and vancomycin-resistant), and some Enterobacteriaceae. According to the World Health Organization (WHO), there is an urgent need for new treatments against these "superbugs". One of the possible approaches in the treatment of these species is the use of antibacterial nanoparticles. After a short overview of nanoparticle usage, mechanisms of action, and methods of synthesis of nanoparticles, emphasis has been placed on the use of silver nanoparticles (AgNPs) to combat the most relevant emerging resistant bacteria. The toxicological aspects of the AgNPs, both in vitro using cell cultures and in vivo have been reviewed. It was found that toxic activity of AgNPs is dependent on dose, size, shape, and electrical charge. The mechanism of action of AgNPs involves interactions at various levels such as plasma membrane, DNA replication, inactivation of protein/enzymes necessary, and formation of reactive oxygen species (ROS) leading to cell death. Researchers do not always agree in their conclusions on the topic and more work is needed in this field before AgNPs can be effectively applied in clinical therapy to combat multi-drug resistant bacteria.

Potential use of machine learning methods in assessment of Fusarium culmorum and Fusariumproliferatum growth and mycotoxin production in treatments with antifungal agents.

Fusarium-controlling fungicides are necessary to limit crop loss. Little is known about the effect of antifungal formulations at sub-lethal doses, and their interaction with abiotic factors, on Fusarium culmorum and F. proliferatum development and on zearalenone and fumonisin biosynthesis, respectively. In the present study different treatments based on sulfur, trifloxystrobin and demethylation inhibitor fungicides (cyproconazole, tebuconazole and prothioconazole) under different environmental conditions, in Maize Extract Medium, are assayed in vitro. Several machine learning methods (neural networks, random forest and extreme gradient boosted trees) have been applied for the first time for modeling growth of F. culmorum and F. proliferatum and zearalenone and fumonisin production, respectively. The most effective treatment was prothioconazole, 250 g/L + tebuconazole, 150 g/L. Effective doses of this formulation for reduction or total growth inhibition ranged as follows ED50 0.49-1.70, ED90 2.57-6.02 and ED100 4.0-8.0 µg/mL, depending on the species, water activity and temperature. Overall, the growth rate and mycotoxin levels in cultures decreased when doses increased. Some treatments in combination with certain aw and temperature values significantly induced toxin production. The extreme gradient boosted tree was the model able to predict growth rate and mycotoxin production with minimum error and maximum R2 value.

Contamination of Wheat, Barley, and Maize Seeds with Toxigenic Fusarium Species and Their Mycotoxins in Tunisia.

BACKGROUND: Fusarium is a worldwide distributed fungal genus. It includes different species pathogenic to cereals among others crops. Some of these species can also produce toxic compounds toward animals and humans. OBJECTIVE: In this work, occurrence of fumonisins B1+B2, zearalenone, type A trichothecenes (T-2 and HT-2 toxins), and type B trichothecenes (deoxynivalenol[DON] and nivalenol[NIV]) was studied in 65 samples of stored and freshly harvested wheat, barley, and maize collected in Tunisia. METHODS: Mycotoxins analyses were performed by using gas chromatography for type B trichothecenes and HPLC for other mycotoxins. Obtained results were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction (PCR). RESULTS: Fumonisins occurred in 20.83% of wheat, 40% of barley, and 57.14% of maize samples, at levels exceeding European limits and suggesting a risk in Tunisian cereals, especially maize. Zearalenone, DON, NIV, and T-2+HT-2 toxins were detected at lower values in only wheat and barley samples. PCR protocols showed the predominance of F. verticillioides especially in maize, and occurrence of F. equiseti and F. graminearum in wheat and barley, and F. proliferatum in only two maize samples. A very consistent correlation was found between the detection of F. verticillioides and the contamination by fumonisins, as well as between the presence of F. graminearum and the contamination by zearalenone, DON, and NIV in the analyzed cereals. CONCLUSIONS: Consequently, the detection of Fusarium species with the current PCR assays strategy in wheat, barley, and maize grains may be considered predictive of their potential mycotoxin risk in these matrices. HIGHLIGHTS: This work is the first to report information on the occurrence of fumonisins, trichothecene, and ZEN, together with their potentially producing Fusarium species in wheat, barley, and maize in Tunisia. The high level of fumonisins in cereals, especially maize, stresses the importance of the control and the regularization of these mycotoxins for food safety.

Machine learning approach for predicting Fusarium culmorum and F. proliferatum growth and mycotoxin production in treatments with ethylene-vinyl alcohol copolymer films containing pure components of essential oils.

Fusarium culmorum and F. proliferatum can grow and produce, respectively, zearalenone (ZEA) and fumonisins (FUM) in different points of the food chain. Application of antifungal chemicals to control these fungi and mycotoxins increases the risk of toxic residues in foods and feeds, and induces fungal resistances. In this study, a new and multidisciplinary approach based on the use of bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing pure components of essential oils (EOCs) and machine learning (ML) methods is evaluated. Bioactive EVOH-EOC films were made incorporating cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG) or linalool (LIN). Several ML methods (neural networks, random forests and extreme gradient boosted trees) and multiple linear regression (MLR) were applied and compared for modeling fungal growth and toxin production under different water activity (aw) (0.96 and 0.99) and temperature (20 and 28 °C) regimes. The effective doses to reduce fungal growth rate (GR) by 50, 90 and 100% (ED50, ED90, and ED100) of EOCs in EVOH films were in the ranges 200 to >3330, 450 to >3330, and 660 to >3330 µg/fungal culture (25 g of partly milled maize kernels in Petri dish), respectively, depending on the EOC, aw and temperature. The type of EVOH-EOC film and EOC doses significantly affected GR in both species and ZEA and FUM production. Temperature also affected GR and aw only affected GR and FUM production of F. proliferatum. EVOH-CIT was the most effective film against both species and ZEA and FUM production. Usually, when the EOC levels increased, GR and mycotoxin levels in the medium decreased although some treatments in combination with certain aw and temperature values induced ZEA production. Random forest models predicted the GR of F. culmorum and F. proliferatum and ZEA and FUM production better than neural networks or extreme gradient boosted trees. The MLR mode provided the worst performance. This is the first approach on the ML potential in the study of the impact that bioactive EVOH films containing EOCs and environmental conditions have on F. culmorum and F. proliferatum growth and on ZEA and FUM production. The results suggest that these innovative packaging systems in combination with ML methods can be promising tools in the prediction and control of the risks associated with these toxigenic fungi and mycotoxins in food.

Antifungal effect of engineered silver nanoparticles on phytopathogenic and toxigenic Fusarium spp. and their impact on mycotoxin accumulation.

Cereal grains are essential ingredient in food, feed and industrial processing. One of the major causes of cereal spoilage and mycotoxin contamination is the presence of toxigenic Fusarium spp. Nanoparticles have immense applications in agriculture, nutrition, medicine or health but their possible impact on the management of toxigenic fungi and mycotoxins have been very little explored. In this report, the potential of silver nanoparticles (AgNPs) (size 14-100 nm) against the major toxigenic Fusarium spp. affecting crops and their effect on mycotoxin accumulation is evaluated for the first time. The studied Fusarium spp. (and associated mycotoxins) were F. graminearum and F. culmorum (deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone), F. sporotrichioides and F. langsethiae (T-2 and HT-2 toxins), F. poae (nivalenol), F. verticillioides and F. proliferatum (fumonisins B1 and B2) and F. oxysporum (mycotoxins no detected). The factors fungal species, AgNP dose (range 2-45 µg/mL), exposure time (range 2-30 h) and their interactions significantly influence spore viability, lag period and growth rate (GR) in subsequent cultures in maize-based medium (MBM) of all the studied species. The effective lethal doses (ED50, ED90 and ED100) to control spore viability and GR were in the range 1->45 µg/mL depending on the remaining factors. At high exposure times (20-30 h), the three effective doses ranged 1-30 µg/mL for all the studied species. At the end of the incubation period (10 days) mycotoxin levels in MBM cultures inoculated with fungal spores from treatments were strongly related with the size reached by the colony at that time. None of the treatments produced stimulation in conidia germination, GR or mycotoxin biosynthesis with respect to controls. Thus, the antifungal effect of the assayed AgNPs against the tested Fusarium spp. suggests that AgNPs could be a new antifungal ingredient in bioactive polymers (paints, films or coating) likely to be implemented in the agro-food sector for controlling these important toxigenic Fusarium spp. and their main associated mycotoxins.

Determination of multiple mycotoxins in feedstuffs by combined use of UPLC-MS/MS and UPLC-QTOF-MS.

In this report, a UPLC-ESI-MS/MS method for the simultaneous determination of aflatoxins, ochratoxin A, zearalenone, deoxynivalenol, fumonisins, T-2 and HT-2 toxins, fusarenone X, diacetoxyscirpenol, and 3- and 15-acetyldeoxynivalenol in feedstuffs was developed. A quadrupole-time-of-flight mass spectrometer detector (QTOF-MS) operating in full scan mode was combined with the UPLC-ESI-MS/MS system to confirm the identity of detected mycotoxins and to identify other possible microbial metabolites occurring in samples. Sixty-two feed samples from the Spanish market were analyzed. Extraction of metabolites was carried out with acetonitrile-water-formic acid (80:19:1, v/v/v). Method detection and quantification limits and performance criteria set by Commission Regulation (EC) No 401/2006 were fulfilled. Relatively high levels of the main regulated mycotoxins and presence of non-regulated mycotoxins in feed samples were found. This is the first study in which mycotoxins and other microbial metabolites occurring in feed are studied using a UPLC-QTOF-MS system being therefore a reference report.

Electrochemical identification of toxigenic fungal species using solid-state voltammetry strategies.

An electrochemical methodology for the characterization of mycotoxin-producing fungal species from the genera Aspergillus and Fusarium using solid-state voltammetry is described. Upon attachment of fungal colony microsamples to glassy carbon electrodes in contact with aqueous acetate buffer, characteristic voltammetric signals mainly associated to the oxidation of polyphenolic metabolites are recorded. The possibility of fungi-localized electrochemical processes was assessed by means of electron microscopy and field emission scanning electrochemical microscopy coupled to the application of oxidative potential inputs. Using pattern recognition methods, the determined voltammetric profiles were able to discriminate between mycotoxin-producing fungi from different sections and to identify selected toxigenic species of the Aspergillus and Fusarium genera isolated from grapes and cereals.

Risk management of ochratoxigenic fungi and ochratoxin A in maize grains by bioactive EVOH films containing individual components of some essential oils.

Aspergillus steynii and Aspergillus tubingensis are possibly the main ochratoxin A (OTA) producing species in Aspergillus section Circumdati and section Nigri, respectively. OTA is a potent nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic and immunosuppressive compound being cereals the first source of OTA in the diet. In this study bioactive ethylene-vinyl alcohol copolymer (EVOH) films containing cinnamaldehyde (CINHO), linalool (LIN), isoeugenol (IEG) or citral (CIT) which are major components of some plant essential oils (EOs) were produced and tested against A. steynii and A. tubingensis growth and OTA production in partly milled maize grains. Due to the favourable safety profile, these bioactive compounds are considered in the category "GRAS". The study was carried out under different water activity (0.96 and 0.99 aw), and temperature (24 and 32 °C) conditions. ANOVA showed that class of film, fungal species, aw and temperature and their interactions significantly affected growth rates (GR), ED50 and ED90 and the doses for total fungal growth inhibition and OTA production. The most effective EVOH films against both species were those containing CINHO. ED50, ED90 and doses for total growth and OTA inhibition were 165-405, 297-614, 333-666 µg of EVOH-CINHO/plate (25 g of maize grains), respectively, depending on environmental conditions. The least efficient were EVOH-LIN films. ED50, ED90 and doses for total growth and OTA inhibition were 2800->3330, >3330 and >3330 µg of EVOH-LIN/plate (25 g of maize grains), respectively. The effectiveness of the bioactive films increased with increasing doses. Overall, A. tubingensis was less sensitive to treatments than A. steynii. Depending on the species, aw and temperature affected GR and OTA production in a different way. In A. steynii cultures, optimal growth occurred at 0.96 aw and 32 °C while optimal OTA production happened at 0.99 aw and 32 °C. In A. tubingensis cultures optimal growth happened at 0.99 aw and 32 °C, although the best conditions for OTA production were 0.99 aw and 24 °C. Thus, these species can be very competitive in warm climates and storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study and applied in vapour phase, can be potent antifungal agents against A. steynii and A. tubingensis and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films containing individual components of EOs have on the growth of these ochratoxigenic fungi and on OTA production in maize grains.

Selected plant essential oils and their main active components, a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food.

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10-1000 mg l-1 were assayed and the effective doses for 50 (ED50) and 90% (ED90) growth inhibition were determined. The ED50 of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49-52.6, 98-145, 152-505, 295-560 mg l-1 and against A. parasiticus in the ranges 46-55.5, 101-175, 260-425 and 490-675 mg l-1, respectively, depending on environmental conditions. In A. flavus treatments ED90 were in the ranges 89.7-90.5, 770-860 and 820->1000 mg l-1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89-91, 855->1000 and 900->1000 mg l-1, respectively. ED90 values for oregano EO against both species were >1000 mg l-1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 aw. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.

Impact of bioactive packaging systems based on EVOH films and essential oils in the control of aflatoxigenic fungi and aflatoxin production in maize.

Aspergillus flavus and A. parasiticus are the most common fungal species associated with aflatoxin (AF) contamination of cereals, especially maize, and other agricultural commodities. AFB1, the most frequent and toxic metabolite, is a powerful hepatotoxic, teratogenic and mutagenic compound. Effective strategies to control these fungal species and AFs in food and feed are required. Active packaging film containing essential oils (EO) is one of the most innovative food packaging concepts. In this study, ethylene-vinyl alcohol (EVOH) copolymer films incorporating EO from Origanum vulgare (ORE), Cinnamomum zeylanicum (CIN) or their major active constituents, carvacrol (CAR) and cinnamaldehyde (CINHO), respectively, were developed and assayed to control growth of A. flavus and A. parasiticus and AF production in maize grains under different aw and temperature regimens. EO doses assayed in cultures were in the range 0.25-4.0mg/Petri dish. The factors aw, temperature, type of EVOH-EO film and fungal species significantly influenced the ED50 values of all assayed films. Growth rate (GR) of both species was usually higher at 0.99 than at 0.96 aw and at 37°C than at 25°C. However, the contrary was found with regard to AF production. The order of efficacy of EVOH-EO films to control growth of both species and AF production was EVOH-CINHO>EVOH-CAR>EVOH-ORE>EVOH-CIN. The effective dose (ED50) (mg EO/plate) for EVOH-CINHO and EVOH-CIN films against A. flavus were in the ranges of 0.125 and 2.475-3.500 and against A. parasiticus in the ranges of 0.121-0.133 and 2.275-3.625, respectively. Under the assayed conditions, the ED90 for EVOH-CINHO film were 0.22-0.23mg/plate for both species. It was the most effective bioactive film to control fungal growth (vapour phase) and AF production, regardless of aw and temperature. This is the first study about the impact that interacting environmental conditions and bioactive EVOH-CINHO, EVOH-ORE, EVOH-CIN EVOH-CAR films have on the growth of aflatoxigenic fungi and on AF production in maize grains.

Assessment of azole fungicides as a tool to control growth of Aspergillus flavus and aflatoxin B1 and B2 production in maize.

Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract-sucrose (YES) agar and in maize kernels under different water activities (aw) and temperatures. Aflatoxins B1 and B2 were determined by LC with fluorescence detection and post-column derivatisation of AFB1. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB1 and AFB2 production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at the combination 37ºC/0.95 aw; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 aw, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l-1 prochloraz or 5.0 mg l-1 tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium.

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED50 and ED90 values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED50 and ED90 values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp.

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.

Determination of type A and type B trichothecenes in paprika and chili pepper using LC-triple quadrupole-MS and GC-ECD.

There is a need to develop sensitive and accurate analytical methods for determining deoxynivalenol (DON), HT-2 toxin and T-2 toxin in paprika to properly assess the relevant risk of human exposure. An optimized analytical method for determination of HT-2 toxin and T-2 toxin using capillary gas chromatography with electron capture detection and another method for determination of DON by liquid chromatography-mass spectrometry in paprika was developed. The method for determination of HT-2 toxin and T-2 toxin that gave the best recoveries involved extraction of the sample with acetonitrile-water (84:16, v/v), clean-up by solid-phase extraction on a cartridge made of different sorbent materials followed by a further clean-up in immunoaffinity column that was specific for the two toxins. The solvent was changed and the eluate was derivatized with pentafluoropropionic anhydride and injected into the GC system. The limits of detection (LOD) for T-2 and HT-2 toxins were 7 and 3 µg/kg, respectively, and the recovery rates for paprika spiked with 1000 µg toxin/kg were 71.1% and 80.1% for HT-2 and T-2 toxins, respectively. For DON determination, the optimized method consisted of extraction with acetonitrile-water (84:16, v/v) solution followed by a solid-phase extraction clean-up process in a cartridge made of different sorbent compounds. After solvent evaporation in N(2) stream, the residue was dissolved and DON was separated and determined by LC-MS/MS. The LOD for this method was 14 µg DON/kg paprika sample and the DON recovery rate was 86.8%.

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium.

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 a(w). Mancozeb at 40 mg l(-1 )inhibited fungal growth and provided lag phases >24 days at 10-20 mg l(-1) and 15°C. OTA was observed only at 25°C and doses <10 mg l(-1). At 15°C, copper oxychloride proved inhibitory at 800 mg l(-1), while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5-8 g l(-1) (at 15°C) while at 25°C growth took place even at 8 g l(-1), although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb > copper oxychloride > sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.

Optimization of clean-up procedure for patulin determination in apple juice and apple purees by liquid chromatography.

Patulin (PAT) is a mycotoxin produced in fruits, mainly in apples, by several fungal species that can be carried into industrial apple juice by-products during factory processing. An analytical method for determination of PAT in apple juice and another one for determination of this compound in apple purees and apple compotes by liquid chromatography are proposed in the present paper. These methods have better precision and sensitivity than previously reported methods and focus mainly on extraction and clean-up. To accomplish analytical methods with higher accuracy, lower limits of detection and simpler procedures for application in quality control of the goods, different extraction and clean-up procedures for PAT were comparatively studied. PAT recoveries in apple juice spiked with 1.0mg PAT/kg varied between 52.3% and 81.0%. The highest PAT recovery in apple puree spiked with 0.1mg PAT/kg was 82.9%. Addition of NaH(2)PO(4) during the extraction phase here reported for the first time has the advantage of keeping the pH slightly acidic, thus avoiding PAT degradation.

Influence of nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains of Aspergillus spp. isolated from grapes.

This work studies the influence of nitrogen and carbon source on ochratoxin A production by three Aspergillus isolates A. ochraceus (Aso2), A. carbonarius (Ac25) and A. tubingensis (Bo66), all isolated from grapes. A basal medium (0.01 g/l FeSO4.7H2O, 0.5 g/l MgSO4.7H2O, 0.5 g/l Na2HPO4.2H2O, 1.0 g/l KCl) was prepared. This medium was supplemented with different nitrogen sources, both inorganic [(NH4)3PO(4), 0.3 g/l plus NH4NO3, 0.2 g/l] and organic (histidine, proline, arginine, phenylalanine, tryptophan or tyrosine) at two concentrations (0.05 g/l or 0.3 g/l), and different carbon sources (sucrose, glucose, maltose, arabinose or fructose) at three concentrations (10 g/l, 50 g/l or 150 g/l). A medium with sucrose (18 g/l) and glucose (1 g/l) was also tested. After a 10-day incubation period at 25 degrees C the highest levels of OTA (44.0 ng/ml, 13.5 ng/ml and 0.49 ng/ml for A. ochraceus, A. carbonarius and A. tubingensis, respectively) were obtained in the cultures containing 150 g/l of arabinose and 0.05 g/l of phenylalanine. Analysis of variance of the data showed that there were significant differences (p-value 0.05) among the OTA levels in the cultures with regard to carbon source and isolate. No significant differences were detected in OTA production regarding nitrogen source, although 0.05 g/l of phenylalanine generally favoured OTA production in the cultures of the three isolates. The dynamics of toxin production in the cultures of each isolate using the optimized basal medium supplemented with 0.05 g/l of phenylalanine and 150 g/l of arabinose for a period of 42 days at 25 degrees C was also studied. The maximum level of OTA was detected on the 3rd day of incubation in A. tubingensis cultures and on the 35th and 43(rd) days of incubation in A. ochraceus and A. carbonarius, respectively. This is the first study in which defined media have been used to assess the influence of carbon and nitrogen sources on OTA production by isolates of OTA-producing species isolated from grapes and to analyse the dynamics of toxin production in these species in a defined culture medium. This optimized medium for OTA production is being used in current studies aimed at elucidating its biosynthetic pathway.

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes.

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.

An overview of ochratoxin A in beer and wine.

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.